As far as I am aware, no. Annexin V-binding is calcium-dependent, hence the buffer.

Have you tried backing up your results with alternative apoptosis assays such as Caspase 3 or TUNEL?

If the annexin buffer is a problem, I would co-stained PI and activated (cleaved) caspase-3. There is a good antibody I could let you know about if you don't have one that you prefer. While caspase 3 activation is a great hallmark for apoptosis as it detects both early and late apoptotic cells (annexin V is a hallmark of late apoptosis stage and misses the ones in early stages of apoptosis), it misses cells dying by other mechanisms. PI staining would let you know if that is happening. That is, if other forms of cell death different from apoptosis are happening in your population. So I recommend you to go for double staining with PI (or other DNA stain) and activated caspase-3. Good luck and let me know if you have further questions.

Best

Daniel

Although there are a number of assays for detection of apoptosis, annexin staining should work. Annexin-PI double staining gives a good idea about apoptosis/other mode of cell death.  It needs a bit standardization, if you have not done it before.  Can you elaborate why you need to omit the annexin buffer?

Thanks all for your answers. I know about caspases activation and TUNEL. I am not trying to omit annexin buffer. Last time i go for the experiment with operetta (high content imaging analysis) i didn't use annexin binding buffer. When i got images or the data from the Harmony software, it looked like the cells didn't took annexin. But they stained with PI. So it looked like PI doesnt need anything to bind with DNA and RNA (both nuclear and cytoplasmic). My confusion is, whether FITC-annexin V will bind to the translocated membrane phosphotydylserin without annexin binding buffer. According to my result my answer is NO. What you people think?

call me 01797320499

The classic Annexin V Binding buffer has 150 mM NaCl, 10 mM HEPES pH 7.4, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2. (Koopman, G et al., Blood 84: 1415, 1994). Without the Ca2+ and Mg2+ ions there will be no binding to PS. Also EDTA or EGTA cannot be in your buffer to get binding to PS. If you are using PBS or HBSS for your cells you could add 1 mM MgCl2 and 1.8 mM CaCl2 to these buffers.

Thanks Ewan M Tytler. It looks convincing. Let me try. I will let you know the update.

i wonder what time window is suitable to detect apoptotic cells using HCS, assuming apoptosis/cell death will cause cell detachment which will cause cell of interest (apoptotic) loss !!